Toxicity of Diazinon 60 EC for Cyprinus carpio and Poecilia reticulata

Diazinon 60 EC (chemical insecticide, organophosphate, active substance diazinon at a concentration of 600 g l⁻¹) is used in fish-farming in well-founded cases as a biocide to suppress excessive propagation of coarse Daphnia zooplankton. At the concentration of 10 μg l⁻¹ (i.e., 100 g per 1 ha at a mean depth of the pond of 1 m), Diazinon 60 EC can highly selectively eliminate Daphnia zooplankton, causes no harm to fish, and relatively quickly decomposes in the aquatic environment. This has been corroborated by results of acute toxicity tests in fish: the 96 h LC50 is 3 mg l⁻¹ for the guppy (Poecilia reticulata) and 10–25 mg l⁻¹ for the common carp (Cyprinus carpio). As demonstrated by embryonic and larval tests of toxicity for the common carp (C. carpio), Diazinon 60 EC at the concentration of 10 μg l⁻¹ causes no harm to the tested, individuals even in these sensitive (critical) stages of ontogeny.     

Effect of light intensity and photoperiod on biomass and fatty acid composition of the microalgae, Chlorella vulgaris

The effects of irradiance and photoperiod on the biomass and fatty acid (FA) composition of Chlorella vulgaris were examined in the exponential growth phase. Results indicated significant differences in biomass and FA at different intensities and photoperiods. Maximum biomass (2.05 ± 0.1 g l⁻¹) was at 62.5 μmol photons m⁻² s⁻¹ and 16:8 h light/dark photoperiod. FA composition changed considerably in different light regimes; the maximum percentage of total saturated (SFA) (33.38%) was recorded at 100 μmol photons m⁻² s⁻¹ and 16:8 h photoperiod, while monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids decreased with increasing irradiance and light duration. The maximum percentage of total MUFA (15.93%) and PUFA (27.40%) was recorded at 37.5 μmol photons m⁻² s⁻¹ and 8:16 h photoperiod.     

Biochemical and histological changes in the brain tissue of spotted murrel, <Emphasis Type="Italic">Channa punctatus</Emphasis> (Bloch), exposed to endosulfan

The present experiment was conducted to establish the relationship between selected physiological parameters and histological responses of Channa punctatus brain tissue to endosulfan exposure. The fish (35.6 ± 0.7 g) was exposed to sublethal endosulfan concentration (8.1 μg l⁻¹) for a period of 12, 24, 36, 48, 72, and 96 h. Results showed that brain glucose level increased significantly after exposure, indicating a hyperglycemic state of the fish. Brain vitamin C level decreased with an increase in the exposure time. Acetylcholine esterase and adenosine triphosphatase enzyme activities also showed a significant reduction upon endosulfan exposure. Brain histopathology after 96 h endosulfan exposure showed that the apical lobe of the cerebrum (the only portion examined) had mild necrosis. Focal area of gliosis could be seen in the cerebrum, which were absent in the control fish. The results indicate that exposure of sublethal concentration of endosulfan to C. punctatus may have a direct effect on the histology of the fish's brain tissue, thereby affecting its metabolism.     

Plasma biochemical responses of the omnivorous crucian carp (<Emphasis Type="Italic">Carassius auratus</Emphasis>) to crude cyanobacterial extracts

Healthy crucian carp (Carassius auratus) were treated by intraperitoneal (i.p.) injection of crude cyanobacterial extracts at two doses, 50 and 200 μg MC-LR equiv kg⁻¹ BW. High mortality (100%) was observed within 60 h post injection in the high-dose group. In the treated fish, activities of four plasma enzymes, alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), all showed substantial increases, with both dose and time-dependent effects. These increases of enzyme activity indicate severe impairment occurred in the liver of crucian carp over time. Plasma concentrations of energy-related biomolecules including glucose (GLU), cholesterol (CHO), triglyceride (TG), and total protein (TP) showed marked changes in the high-dose group, possibly a nutritional imbalance correlated with the liver injury caused by intraperitoneal exposure to crude cyanobacterial extracts.     

Acute and chronic effects of endosulfan on the haemato-immunological and histopathological responses of a threatened freshwater fish, spotted murrel, Channa punctatus

Two experiments, one short-term and one long-term, were conducted to elucidate the acute and chronic effects, respectively, of endosulfan exposure on the haemato-immunological and histopathological responses of Channa puncatatus. In the short-term study, fish were exposed to sublethal endosulfan (8.1 μg l⁻¹) for 12, 24, 36, 48, 72 and 96 h. In the long-term study, fish were fed with normal diet and simultaneously either exposed to endosulfan (1.2 μg l⁻¹) for 90 days or not. Results showed that the ascorbic acid levels in both the liver and the muscle decreased significantly (P < 0.05) by acute and chronic endosulfan exposure. The haemoglobin (Hb) level reduced significantly (P < 0.05) by 15.5% within 12 h of acute endosulfan exposure, further decreased by 25.8% after 24 h of exposure, however, thereafter the values increased and at the end of 72 h returned to normal levels. Almost similar trend was observed for the erythrocyte (RBC) count. The WBC count and the nitroblue tetrazolium (NBT) value showed a general increasing trend with increase in the duration of acute endosulfan exposure. The chronic exposure of C. punctatus to endosulfan significantly (P < 0.05) lowered the Hb level, RBC and WBC counts, NBT reduction value and the plasma parameters such as plasma protein, albumin (A) and globulin (G) compared with that of the control (except for A/G ratio). Endosulfan exposure also severely altered the liver histological structure. Overall results indicated that both short-term acute and long-term chronic endosulfan exposure had a significant impact on the haemato-immunological parameters and tissue histopathology of C. punctatus.     

Influence of alginic acid and fucoidan on the immune responses of head kidney leukocytes in cod

The present study investigated the immunomodulatory activities of alginic acid and fucoidan, both derived from brown seaweeds, on selected cellular immune responses and antibacterial activity of head kidney (HK) leukocytes of cod, Gadus morhua. Primary cultures of HK leukocytes were incubated with either 10 or 100 μg ml⁻¹ of the substances and the effects on respiratory burst, cellular proliferation, acid and alkaline phosphatase activity and cellular myeloperoxidase were measured at 3- and 24-h post-incubation. The antibacterial activity of the supernatants collected from the cell cultures incubated with 100 μg ml⁻¹ of the substances were tested against Vibrio anguillarum and Aeromonas salmonicida. Respiratory burst was significantly elevated in cells incubated with either alginic acid or fucoidan in a dose-dependent manner. Incubation with a higher dose of alginic acid and fucoidan resulted in lower cellular proliferation at 3- and 24-h, respectively. Both acid and alkaline phosphatase activities of HK leukocytes were not significantly modulated, except for a slight elevation of acid phosphatase in cells incubated with 100 μg ml⁻¹ of alginic acid for 24-h. Fucoidan, but not alginic acid significantly increased cellular myeloperoxidase activity at a concentration of 100 μg ml⁻¹. The growth of the bacteria in both the treated and control supernatants was significantly lower than what was observed in the bacterial culture medium. However, the supernatants from the treated cells had significantly higher bacterial growth compared with supernatants of the control cells. Taken together, these results showed that at the tested concentrations, both alginic acid and fucoidan are able to differentially stimulate some cellular immune responses of cod HK leukocytes in vitro and the respiratory burst activity was significantly stimulated by these brown algal derivatives. These substances could be tested as potential immunostimulants in future in vivo studies.     

Application of synthetic hormone LHRH-A<Subscript>2</Subscript> on the artificial propagation of Persian sturgeon <Emphasis Type="Italic">Acipenser persicus</Emphasis>

For studying the effect of LHRH-A₂ hormone on the induction of final maturation and ovulation of Persian sturgeon, 71 matured females and 20 matured males were used. Five groups of breeders were injected with sturgeon pituitary gland hormone (50 mg per fish) and LHRH-A₂ in dosages of 3.5, 7, 8 and 10 μg kg⁻¹ for females and 3 and 5 μg kg⁻¹ for males. Results showed that LHRH-A₂ successfully induced final maturation and ovulation in females, and there was no significant difference between five groups of breeders in ovulation proportion, fertilization rate, survival rate of incubation, survival of yolk-sac absorption period and active feeding period of larvae. It can be concluded that the LHRH-A₂ is a proper alternative for pituitary gland hormone in artificial propagation of Persian sturgeon.     

Cod CGRP and tachykinins in coeliac artery innervation of the Atlantic cod, <Emphasis Type="Italic">Gadus morhua</Emphasis>: presence and vasoactivity

The presence and vasoactive effects of native calcitonin gene-related peptide (CGRP), substance P (SP), and neurokinin A (NKA) were studied on isolated small branches of the coeliac artery from Atlantic cod, Gadus morhua, using immunohistochemistry and myograph recordings, respectively. Immunohistochemistry revealed nerve fibers containing CGRP- and SP/NKA-like material running along the wall of the arteries. CGRP induced vasorelaxation of precontracted arteries with a pD₂ value of 8.54 ± 0.17. Relaxation to CGRP (10⁻⁸ M) was unaffected by l-NAME (3 × 10⁻⁴ M) and indomethacin (10⁻⁶ M) suggesting no involvement of nitric oxide or prostaglandins in the CGRP-induced relaxation. SP and NKA (from 10⁻¹⁰ to 3 × 10⁻⁷ M) contracted the unstimulated arteries at concentrations from 10⁻⁸ M and above in 42% and 33%, respectively, of the vessels. It is concluded that the innervation of the cod celiac artery includes nerves expressing CGRP-like and tachykinin-like material, and that a vasodilatory response to CGRP is highly conserved amongst vertebrates while the response to tachykinins is more variable.     

Evaluation of a 40 day Assay for Testing Endocrine Disrupters: Effects of an Anti-Estrogen and an Aromatase Inhibitor on Sex Ratio and Vitellogenin Concentrations in Juvenile Zebrafish (Danio rerio)

In the present study, the effects of the anti-estrogen ZM 189,156 and the aromatase inhibitor fadrozole were evaluated in a 40-day juvenile assay developed for screening of endocrine disrupting chemicals. Juvenile zebrafish were exposed from 20 to 60 days post hatch (dph) to ZM 189, 154 (100 μg l⁻¹ and 200 μg l⁻¹) and fadrozole (10, 32, and 100 μg l⁻¹). VTG concentrations were measured at 38 dph by the use of a direct non-competitive sandwich ELISA and sex ratios were determined at 60 dph by histological examination of the gonads. A small but significant increase in VTG concentrations was observed in fish exposed to ZM 189, 156 (100 and 200 μg l⁻¹) and fadrozole (10 and 100 μg l⁻¹) compared to control groups. In fish exposed to ZM 189, 156 and fadrozole, the percentage of females declined and the number of undifferentiated fish increased. These findings show that exposure of juvenile zebrafish to an aromatase inhibitor or an anti-estrogen during early development inhibits differentiation and development of female gonads. The data presented, furthermore, show that the 40-day juvenile assay may be suitable for screening endocrine disrupting chemicals acting as anti-estrogens and aromatase inhibitors.     

Ammonia autointoxication of common carp: case studies

Three case studies of ammonia autointoxication of the common carp (Cyprinus carpio L.) are described. In the first case, carp yearlings with a full digestive tract were transferred during the growing period from pond water (22°C) to tap water (17°C). In the second case, marketable carp were transferred from the fishing ground of a pond (18°C) to a storage pond (10–12°C). Harvest was performed in late September when the fish were still ingesting natural feed. In the third case, marketable carp after storage for 1 month were transferred to storage ponds with markedly lower water temperature. Stress because of harvest and handling also occurred in this case. In all cases, highly increased concentrations of ammonia were found in the blood plasma of the fish (mean ± SD 1760 ± 350 μmol L⁻¹ in the first case, 870 ± 540 μmol L⁻¹ in the second case, and 880 ± 150 μmol L⁻¹ in the third case). Highly congested, dark-red coloured, oedematous gills were observed for all specimens. We can avoid similar cases of ammonia autointoxication by protection of fish from sudden changes of temperature during rearing, harvesting, and handling.     

Evaluation of three phytoplankton species as food for the pearl oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis>

In the pearl cultivation farms of the Ehime Prefecture, Japan, mass mortalities of the pearl oyster Pinctada fucata have occurred since 1994. The occurrences of mass mortality roughly coincided with a shift of the dominant phytoplankton from Skeletonema and Chaetoceros to Chaetoceros and Nitzschia all of which belong to Bacillariophyceae. Hence, we evaluated Nitzschia, together with Chaetoceros and Isocrysis, as food for the oyster. Wet weights, lengths, widths, glycogen contents, and growth rates in terms of wet weight of the oysters in all the feeding treatments were significantly higher than those in the non-feeding treatment. The highest glycogen content (2.34%) and growth rate (2.21 g month⁻¹) were found in the Chaetoceros treatment. Growth rate in the Isocrysis treatment (1.63 g month⁻¹) was also high, although glycogen content in this treatment (0.41%) was low. In the Nitzschia treatment, growth rate of the oyster (0.94 g month⁻¹) was the lowest and glycogen content (0.83%) was also low relative to that in the Chaetoceros treatment. Chlorophyll a concentration in fecal pellets was lowest in the Nitzschia treatment (<2.7 μg mg⁻¹), suggesting more complete digestion of Nitzschia by the oyster. Thus, Nitzschia was edible and digestible but not assimilated by P. fucata. We propose the following scenario for the relationship between Nitzschia dominance and mass mortality. When Nitzschia dominates in a culture area, the physiological condition of P. fucata deteriorates due to low assimilation of Nitzschia by the oyster, followed by susceptibility of the oyster to infection by agents lethal to the oyster.     

The effect of praziquantel applied per os on selected haematological and biochemical indices in common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

The aim of the study was to assess the effects of the anthelminthic, praziquantel, on the haematological and biochemical indices of the blood of common carp (Cyprinus carpio L.). Fish were divided into six groups: two groups received 30 mg kg⁻¹ body weight (bw) of praziquantel, and two groups were given 50 mg kg⁻¹ bw of praziquantel mixed into the heat-treated amyloid vehicle. Fish in the remaining two groups were given only the amyloid vehicle and were used as controls. Fish were examined either 24 or 96 h after administration. Praziquantel treatment was characterised by a significantly lower erythrocyte count, haemoglobin level, packed cell volume and total protein at both dose levels after 24 h compared with the controls, but these parameters were similar to the control values at 96 h. The activity of alanine aminotransferase (ALT) was significantly higher after 96 h in the treated groups, which could be attributed to slight hepatocyte damage caused by praziquantel. However, the differences between the treated and the control groups were not great, and we presumed that they were reversible.     

Acute toxicity of ammonia on juvenile cobia (Rachycentron canadum, Linnaeus, 1766) according to the salinity

Juvenile cobia (Rachycentron canadum) (total length 15.16 ± 0.92 cm and weight 19.26 ± 4.5 g) were exposed to different concentrations of ammonia–N (unionized plus ionized ammonia as nitrogen), using the static renewal method at different salinity levels of 5, 20, and 35‰ at pH 8.1 and 25°C. The 24, 48, 72, 96 h LC₅₀ values of ammonia–N for R. canadum juveniles were 60.28, 48.57, 37.42, 22.73 mg l⁻¹ at 35‰; 51.25, 43.63, 28.17, 19.05 mg l⁻¹ at 20‰; and 39.48, 25.31, 19.50, 8.13 mg l⁻¹ at 5‰, respectively. The 24, 48, 72, 96 h LC₅₀ values of NH₃–N (unionized ammonia as nitrogen) were 1.81, 1.46, 1.12, and 0.68 mg l⁻¹ at 35‰; 1.75, 1.49, 0.96, and 0.65 mg l⁻¹ at 20‰; and 1.52, 0.97, 0.71, and 0.31 mg l⁻¹ at 5‰, respectively. As the salinity decreased from 35 to 5‰, susceptibility of ammonia–N increased by 34.5, 47.88, 50.56, and 64.23% after 24, 48, 72, and 96 h exposure, respectively. Furthermore, we found that exposure of fish to ammonia–N caused an increase in oxygen consumption of 129.1, 157.5, and 192% and a decrease in the ammonia excretion level of 53.4, 38.2, and 23.3% with respect to the control.     

Purification and characterization of α<Subscript>1</Subscript>-proteinase inhibitor and antithrombin III: major serpins of rainbow trout (<Emphasis Type="Italic">Oncorhynchuss mykiss</Emphasis>) and carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>) blood plasma

The main serine proteinase inhibitors of rainbow trout (Oncorhynchuss mykiss) and common carp (Cyprinus carpio) blood plasma were isolated and purified. The investigated inhibitors, α₁-proteinase inhibitor (α₁-PI) and antithrombin III (AT III), act by forming stable complexes with target proteinases. The association rate constants k _on for the interaction of fish plasma inhibitors with several serine proteinases have been determined: k _on for both carp and rainbow trout α₁-PI were >10⁷ M⁻¹ s⁻¹ for human neutrophil elastase, and in the case of bovine trypsin and chymotrypsin k _on values were 2.0–5.2 × 10⁶ M⁻¹ s⁻¹. Association rate constants k _on for the interaction of carp and rainbow trout AT III with bovine trypsin and thrombin were about 1.3 × 10⁴–7.9 × 10⁵ M⁻¹ s⁻¹ without and >10⁷ M⁻¹ s⁻¹ in presence of heparin; so antithrombins require the presence of heparin to become effective proteinase inhibitors. The high degree of homology of the estimated amino acid sequences of fish inhibitors reactive site loops confirms their similarity with other proteinase inhibitors from the serpin family.     

Acute toxicity of nitrite on tilapia (Oreochromis niloticus) at different external chloride concentrations

Tilapias (Oreochromis niloticus) juveniles (total length 4.9 ± 0.2 cm and weight 1.8 ± 0.2 g) were exposed to several nitrite concentrations (0, 10, 18, 32, 56 and 100 mg l⁻¹) for 96 h, using a semi-static renewal method at chloride levels of 35.0 and 70.0 mg l⁻¹. At the end of the 96-h period, the median lethal concentration (LC₅₀) of nitrite was 28.18 mg l⁻¹ in water with low chloride content (35.0 mg l⁻¹) and 44.67 mg l⁻¹ with high chloride content (70.0 mg l⁻¹, respectively). It indicated that high concentrations of chloride ions could reduce the toxicity of nitrite. During the toxicity experiments, the behaviour and clinical signs of tilapias were also observed. Furthermore, the test of toxic mechanism was designed taking five test concentrations, viz., 5, 10, 15 and 20 mg l⁻¹ and a nitrite-free control. Nitrite exposure produced high levels of methaemoglobin (MHb) but did not seem to cause mortality, as surviving tilapias showed high levels (85.37 ± 2.23 and 53.82 ± 3.44 at 35.0 and 70.0 mg l⁻¹ chloride, respectively). The percentage of MHb exposed to nitrite was significantly higher (P<0.05) than the control (0 mg l⁻¹ nitrite) and increased with the increasing nitrite concentration. However, the percentage of MHb decreased with the increasing chloride concentration.     

Acute effects of benzo[a]pyrene on liver phase I and II enzymes,and DNA damage on sea bream <Emphasis Type="Italic">Sparus aurata</Emphasis>

In the present study biotransformation and detoxification responses to acute exposure to the polycyclic aromatic hydrocarbons benzo[a]pyrene (B[a]P) were investigated in the liver of Sparus aurata (sea bream). Sexually immature gilthead sea bream were treated by intraperitoneal injection of B[a]P (20 mg kg⁻¹) for 6, 12, 24, and 48 h. B[a]P accumulation was quantified in sea bream liver by mean of gas phase chromatography (GPC-MS) after the various exposure periods. The following biological responses were measured: (1) ethoxyresorufin-O-deethylase (EROD) activity, as a phase I biotransformation parameter; (2) liver glutathione S-transferase (GST) activity as a phase II conjugation enzyme. DNA damage was assessed over time using the single-cell gel electrophoresis comet assay. B[a]P bioaccumulation in the liver resulted in a biphasic curve with an increasing uptake up to 5.55 ± 0.67 μg g⁻¹ dry weight after only 6 h exposure and 4.67 ± 0.68 μg g⁻¹ dry weight after 48 h exposure. EROD activity showed a nonsymmetrical bell-shaped kinetic with a maximum at 24 h and lower but significant activities at 12 and 48 h with respect to control animals. Hepatic GST activities were only significant after 48 h exposure. Comet assay showed an increase in liver cells DNA damage with a maximum after 48 h exposure reaching up to 12.17 %DNA in the tail.     

Difference Between Tetrodotoxin and Saxitoxins in Accumulation in Puffer Fish Takifugu rubripes Liver Tissue Slices

In vitro accumulation of tetrodotoxin (TTX) and paralytic shellfish toxin (PST) in tiger puffer fish Takifugu rubripes was investigated using liver tissue slices. When T. rubripes liver slices were incubated with Leibovitz’s L-15 medium containing 0.13 mM TTX at 20 °C in air with saturated humidity, they accumulated 21.5 ± 7.3 μg TTX g⁻¹ liver after the incubation for 12 h and increased to 55.3 ± 8.2 μg TTX g⁻¹ liver at 48 h. In the incubation of T. rubripes liver slices with 0.13 mM PST-containing medium, PST was detected 6.3 ± 0.9 μg g⁻¹ liver at 12 h and reached a plateau thereafter. These results reveal the difference between TTX and PST in accumulation in T. rubripes liver tissue slices. To examine the variation in PST accumulation among fish species, the liver tissue slices from tiger puffer fish T. rubripes, parrot-bass Oplegnathus fasciatus and green ling Hexagrammos otakii were incubated at a concentration of 0.027 mM PST. The toxin contents of 3.0 μg g⁻¹ liver were observed at 8 h regardless of fish species but were not increased subsequently, showing no variety among these three species as to accumulation patterns of PST. It is noted that the tiger puffer fish T. rubripes liver specifically accumulate TTX in preference to PST.     

Effects of stocking density on survival and growth of juvenile tench (<Emphasis Type="Italic">Tinca tinca</Emphasis> L.)

In two 120-day experiments, performed in the laboratory at 22°C, the effects of stocking density on the survival and growth of juvenile tench (Tinca tinca L.) were evaluated. Fish were kept in fibreglass tanks, supplied throughout with flow of artesian water, and fed a dry diet for salmonids, in excess, supplemented with restricted amounts of Artemia nauplii. In the first experiment four-month-old juveniles (0.31 ± 0.04 g and 32.00 ± 1.17 mm TL) were stocked at four densities—0.18, 0.88, 1.05, and 2.10 g l⁻¹. Survival was high (>89%) for all treatments. Final densities ranged between 1.10 g l⁻¹ (significantly lowest) and 10.46 g l⁻¹ (significantly highest). The density increase was significantly higher (611%) for fish stocked at the lowest initial density (0.18 g l⁻¹) than for fish stocked at 0.88, 1.05, and 2.10 g l⁻¹, for which the density increase averaged 457%. In the second experiment, 4.5-month-old juveniles (0.58 ± 0.17 g and 39.54 ± 0.83 mm TL) were stocked at three densities—1.05, 3.00, and 4.00 g l⁻¹. Survival was high (>96%) for all treatments. Final densities ranged between 4.08 and 16.53 g l⁻¹ and were significantly higher for greater initial densities. The density increase was greatest (413%) for fish stocked at the highest density (4 g l⁻¹) and was not significantly different for fish stocked at 1.05 and 3 g l⁻¹. Considering all the densities in the two experiments, for stocking at 4 g l⁻¹ the final density was 15 times higher than that reached after stocking at 0.18 g l⁻¹, without harmful effects on survival and growth. This final density (equivalent to 16.53 kg m⁻³) is in the range recommended for other fish species in this period under intensive conditions.     

Effect of prebiotic xylooligosaccharides on growth performances and digestive enzyme activities of allogynogenetic crucian carp (<Emphasis Type="Italic">Carassius auratus gibelio</Emphasis>)

The effect of prebiotic xylooligosaccharides (XOS) on the growth performance and digestive enzyme activities of the allogynogenetic crucian carp, Carassius auratus gibelio, was investigated. XOS was added to fish basal semi-purified diets at three concentrations by dry feed weight: diet 1, 50 mg kg⁻¹; diet 2, 100 mg kg⁻¹; diet 3, 200 mg kg⁻¹, respectively. Twelve aquaria (n = 20) with three replicates for each treatment group (diets 1–3) and control treated without XOS were used. Weights of all collected carp from each aquarium were determined at the initial phase and at the end of the experiment, and the carp survival was also determined by counting the individuals in each aquarium. After 45 days, there were significant differences (P < 0.05) in the relative gain rate (RGR), and daily weight gain (DWG) of diets 1–3 were compared with the control. However, the survival rate was not affected (P > 0.05) by the dietary treatments. For enzymatic analysis, dissection produced a crude mixture of intestine and hepatopancreas of each segment to measure. The protease activity in the intestine and hepatopancreas content of fish in diet 2 (487.37 ± 20.58 U g⁻¹ and 20.52 ± 1.93 U g⁻¹) were significantly different (P < 0.05) from that in the control (428.13 ± 23.26 U g⁻¹ and 12.81 ± 1.52 U g⁻¹) and diet 3 (428.00 ± 23.78 U g⁻¹ and 14.04 ± 1.59 U g⁻¹). Amylase activity in the intestine was significantly higher for diet 2 compared to diet 1 and the control. As for amylase in the hepatopancreas, assays showed higher activity in diet 2 (P < 0.05) compared to the rest.